Development of a PCR-based method for detection of pathogenic Yersinia enterocolitica in pork Susanne Thisted Lambertz Research and Development Department, National Food Administration, Uppsala, Sweden and Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala Doctoral thesis Swedish University of Agricultural Sciences Uppsala 2005 Acta Universitatis Agriculturae Sueciae 2005:123 ISSN 1652-6880 ISBN 91-576-6922-8 © 2005 Susanne Thisted Lambertz, Uppsala Tryck: SLU Service/Repro, Uppsala 2005 Abstract Thisted Lambertz, S. 2005. Development of a PCR-based method for detection of pathogenic Yersinia enterocolitica in pork. Doctor´s dissertation. ISSN 1652-6880, ISBN 91-576-6922-8 During the last decade, Yersinia enterocolitica has been reported to cause between 500 and 800 cases of human gastroenteritis per year in Sweden. As pigs are the only animals in human food production that regularly harbour the pathogen, pork is probably an important source of infection. Earlier it has only rarely been possible to recover the bacterium from pork, but in the last few years this was made possible by DNA-based technology. In this project, a PCR-based method for the detection of pathogenic Y. enterocolitica in pork was developed. The chromosome-located gene attachment invasion locus (ail) was chosen as the PCR- target. The ail PCR assay was evaluated according to criteria for a standardised PCR-based method set by the European research project FOOD-PCR. In a trial involving 14 European laboratories, the ail PCR assay showed high repeatability and robustness. The complete PCR-based method comprises a sample treatment step prior to the ail PCR assay. The assay consists of either one (single) or two (nested) PCR analyses and an internal amplification control for monitoring false-negative results. The detection limit of the complete (single) PCR method, using inoculated enriched homogenates, was established to 10 cfu or less per gram. An increased sensitivity in the form of a nested PCR was required to enable detection of the bacterium in naturally contaminated pork. This is in practice very important. Finally, for characterisation of isolated strains, a multiplex PCR assay was developed, directed towards four different virulence-associated genes (yst, rfbC, ail and virF). As presence or absence of the four PCR targets was established, the following groups were identified: pathogenic Y. enterocolitica 4/O:3 strains, pathogenic Y. enterocolitica serotypes other than 4/O:3, Y. pseudotuberculosis strains and nonpathogenic strains. The method does not allow for confirmation of the viability of the pathogen, the reason being that the bacterium cannot be isolated by traditional culture. The method can therefore preferably be used where information about viability is not important, for example in studies to identify the critical points during slaughter, important to limit contamination by the bacterium. Keywords: PCR; Yersinia enterocolitica 4/O:3; foodborne pathogen; Sample treatment; buoyant density centrifugation; internal amplification control, pork. Author’s address: Susanne Thisted Lambertz, Division of Microbiology, National Food Administration, P.O. Box 622, SE-751 26 Sweden. To my dear family Contents Appendix, 7 Abbreviations, 8 Introduction, 9 General background, 9 Classification, 9 Y. enterocolitica subtypes, 9 Yersiniosis, 10 Human incidence, 13 Sporadic cases and outbreaks, 14 The virulence plasmid, 14 Biochemical characteristics and detection, 15 A description of pathogenic Y. enterocolitica (4/O:3),16 Presence in the environment (including water), 16 Presence in animals, 16 Presence in food, 17 As a hazard in food, 19 Detection of pathogenic Y. enterocolitica in food, 21 Conventional culture methods, 21 PCR-based methods, 21 PCR targets, 25 Validation of PCR methods, 26 Aims, 28 Materials and methods, 29 Techniques used, 29 Polymerase chain reaction, PCR, 29 Variants of PCR assays, 29 Pulsed-field gel electrophoresis, PFGE, 30 Buoyant density centrifugation, BDC, 30 Collaborative trials (data not published), 30 Within the European Union research project FOOD-PCR, 30 Within the Nordic Committee on Food Analysis, NMKL, 31 General comments on Materials and methods, 32 Bacterial strains, 32 Sample treatment prior to PCR, 32 Bovine serum albumin, BSA, 32 Internal amplification control, IAC, 33 Digest of the ail PCR product (data not published), 33 Terms, 34 Results, 35 Development of two PCR assays, 35 The ail PCR assay, 35 Evaluation of the ail PCR assay, 35 The detection limit of the ail PCR assay (data not published), 35 Digest of the ail PCR product (data not published), 36 The virF PCR assay, 36 Evaluation of the virF PCR assay, 36 Development of the PCR-based method, 36 The pre-PCR sample treatment, BDC, 36 Detection limit of the PCR-based method, 36 Detection in naturally contaminated pork samples, 37 Collaborative trials (data not published), 37 Within the framework of the EU research project FOOD-PCR, 37 Within the framework of the Nordic Committee on Food Analysis NMKL, 38 Discussion, 40 Difficulties in isolation of strains of pathogenic Y. enterocolitica, 40 Buoyant density centrifugation (BDC) as pre-PCR sample treatment, 42 The ail gene as the target molecule for detection, 42 Steps towards a standardised PCR-based method, 44 Detection of pathogenic Y. enterocolitica in pork, 46 PCR used for identification of pathogenic Y. enterocolitica isolates, 47 References, 49 Acknowledgements, 58 Sammanfattning, 60 Appendix Paper I-V This thesis is based on the following papers, which are referred to in the text by their Roman numerals: I. Thisted Lambertz, S., Ballagi-Pordány, A., Nilsson, A., Norberg, P. & Danielsson-Tham, M-L. 1996. A comparison between a PCR method and a conventional culture method for detecting pathogenic Yersinia enterocolitica in food. Journal of Applied Bacteriology 81, 303-308. II. Thisted Lambertz, S., Ballagi-Pordány, A. & Lindqvist R. 1998. A mimic as internal standard to monitor PCR analysis of food-borne pathogens. Letters in Applied Microbiology 26, 9-11. III. Thisted Lambertz, S., Lindqvist, R., Ballagi-Pordány, A. & Danielsson- Tham, M-L. 2000. A combined culture and PCR method for detection of pathogenic Yersinia enterocolitica in food. International Journal of Food Microbiology 57, 63-73. IV. Thisted Lambertz, S. & Danielsson-Tham, M-L. 2005. P IdentificationP and characterization of pathogenic Yersinia enterocolitica by PCR and PFGE. Applied and Environmental Microbioogyl 71, 3674-3681. V. Thisted Lambertz, S., Granath, K., Fredriksson-Ahomaa, M., Johansson, K- E. & Danielsson-Tham, M-L. Evaluation of a combined culture and PCR method for detection of pathogenic Yersinia enterocolitica in food. In manuscript. Papers I, II, III and IV are reproduced with the kind permission of the journals concerned. 7 Abbreviations ail attachment invasion locus API analytical profile index BD buoyant density BDC buoyant density centrifugation BHI brain heart infusion BOS oxalate sorbose broth CFU colony forming units CIN cefsaludin irgasan novobiosin CR-BHO Congo red brain heart infusion agarose DNA deoxyribonucleic acid Escul esculin PFGE pulsed-field gel electrophoresis Pyz pyrazinamidase ISO International Organisation of Standardisation ITC irgasan ticarcillin chlorate KOH potassium hydroxide Lip lipase MRB modified rappaport broth NFA National Food Administration NMKL Nordic Committee on Food Analysis NotI Nocardia otitidis-caviarum, restriction endonuclease PBS phosphate buffer saline PCR polymerase chain reaction REAP restriction enzyme analysis of the plasmid rfbC the O:3-antigen gene Sal salicin SIK Swedish Institute for Food and Biotechnology SMI Swedish Institute for Infectious Disease Control SSDC salmonella shigella agar with deoxycholate Taq Thermus aquaticus Treh trehalose TSB trypton soya broth USDA US Department of Agriculture virF virulence-regulatory factor VP voges-proskauer Xyl xylose yst yersinia heat-stable toxin 8 Introduction General background The genus Yersinia, named in 1944 after the French bacteriologist Alexandre Yersin, comprises eleven species, of which three are human pathogens: Y. enterocolitica, Y. pseudotuberculosis and Y. pestis (Table 1). Y. pestis causes bubonic and pneumonic plague, is transmitted by flea bites or aerosols, and has a narrow range of animal reservoirs and a restricted flea-rodent-flea life cycle. In contrast, Y. enterocolitica and Y. pseudotuberculosis behave like true enteropathogens. They cause intestinal symptoms of moderate intensity, they are transmitted by the faecal-oral route and they spend a part of their life cycle in the environment. The reasons for these dramatic differences in epidemiological characteristics and clinical symptoms remain largely unknown (Carniel, 2003). Classification According to Bergey´s Manual of Systematic Bacteriology, the genus Yersinia belongs to the phylum Proteobacteria, class Gammaproteobacteria, order Enterobacteriales, family Enterobacteriaceae. The genus currently consists of the following species: Y. pseudotuberculosis, Y. pestis, Y. enterocolitica, Y. inter- media, Y. kristensenii, Y. frederiksenii, Y. aldovae, Y. rohdei, Y. mollaretii, Y. bercovieri and Y. ruckeri. The species Y. ruckeri is included in the genus Yersinia but its classification is controversial. Y. pestis and Y. pseudotuberculosis share greater than 90% DNA homology, which suggests that they are different patho- types of the same species. The current species concept is based on two organisms